METHOD FOR PRODUCING L-METHIONINE

Abstract

Provided is a process for the preparation of L-methionine in an enzymatic reaction utilizing dimethyl disulfide (DMDS) a precursor of L-methionine, and an organic reducing compound. In the process, methyl mercaptan can be formed by the enzymatic hydrogenolysis of the DMDS.

Claims

1-15: (canceled)

16. A process for the preparation of L-methionine, comprising: (a) preparing a mixture, comprising: (1) dimethyl disulfide (DMDS), (2) a catalytic amount of an amino acid bearing a thiol group or of a thiol-group-containing peptide, wherein the amino acid bearing a thiol group or the thiol-group-containing peptide may optionally be in the form of the corresponding disulfide, (3) a catalytic amount of a reductase enzyme catalyzing the reduction reaction of a disulfide bridge of the amino acid bearing a thiol group or of the thiol-group-containing peptide, (4) an organic reducing compound in a stoichiometric amount relative to the disulfide, (5) a catalytic amount of a dehydrogenase enzyme catalyzing the reaction for the dehydrogenation of the organic reducing compound, (6) a catalytic amount of a cofactor common to the reductase enzyme and the dehydrogenase enzyme, (b) carrying out an enzymatic reaction to form methyl mercaptan (CH.sub.3SH), (c) adding a precursor of L-methionine and converting the precursor by reacting with the methyl mercaptan formed in (b) to produce L-methionine, and (d) recovering and optionally purifying the L-methionine.

17. The process of claim 16, comprising: (a) preparing a mixture, comprising: (1) dimethyl disulfide (DMDS), (2) a catalytic amount of an amino acid bearing a thiol group or of a thiol-group-containing peptide, wherein the amino acid bearing a thiol group or the thiol-group-containing peptide may be in the form of the corresponding disulfide, (3) a catalytic amount of a reductase enzyme catalyzing the reduction reaction of the disulfide bridge of the amino acid bearing a thiol group or of the thiol-group-containing peptide, and (4) a catalytic amount of NADPH, (b) adding an organic reducing compound in a stoichiometric amount relative to the dimethyl disulfide with a catalytic amount of the dehydrogenase enzyme catalyzing the reaction for the dehydrogenation of the organic reducing compound, (c) carrying out the enzymatic reaction to form methyl mercaptan (CH.sub.3SH), (d) converting an L-methionine precursor with the methyl mercaptan formed in (c) to L-methionine, and (e) recovering and optionally purifying the L-methionine.

18. The process of claim 16, wherein the methyl mercaptan is directly placed in contact with a methionine precursor.

19. The process of claim 16, wherein the organic reducing compound is a hydrogen-donating organic reducing compound bearing a hydroxyl function and is chosen from alcohols, polyols, and sugars.

20. The process of claim 16, wherein the organic reducing compound is chosen from glucose, glucose 6-phosphate, and isopropanol.

21. The process of claim 16, wherein the amino acid bearing a thiol group or the peptide bearing a thiol group is chosen from cysteine, homocysteine, glutathione and thioredoxin.

22. The process of claim 16, wherein the L-methionine precursor is chosen from O-acetyl-L-homoserine and O-succinyl-L-homoserine.

23. The process of claim 16, wherein the methyl mercaptan is directly used, upon leaving the reactor, in the synthesis of the L-methionine.

24. The process of claim 23, comprising: (1) preparing an L-methionine precursor, (2) enzymatically reducing of DMDS in a first reactor with formation of methyl mercaptan leaving the first reactor, (3) enzymatically synthesizing L-methionine in a second reactor with the precursor from (1) and the methyl mercaptan from (2), optionally forming gluconolactone, (4) optionally, recycling the gluconolactone formed in (3) into (1), and (5) recovering and optionally purifying the L-methionine.

25. The process of claim 16, wherein the synthesis of methyl mercaptan from DMDS and the synthesis of L-methionine from the methyl mercaptan are carried out in one and the same reactor.

26. The process of claim 25, comprising: (1) preparing an L-methionine precursor by bacterial fermentation of glucose, (2) enzymatically reducing DMDS in a first reactor with in situ formation of methyl mercaptan and concomitant enzymatic synthesis of L-methionine in the same reactor with the L-methionine precursor obtained in (1), optionally forming gluconolactone, (3) optionally, recycling the gluconolactone formed in (2) into (1), and (4) recovering and optionally purifying the L-methionine.

27. The process of claim 16, which is carried out batchwise or continuously.

28. The process of claim 16, wherein the organic reducing compound/DMDS molar ratio varies from 0.01 to 100.

29. The process of claim 16, wherein the DMDS/L-methionine precursor molar ratio is between 0.1 and 10.

30. The process of claim 16, wherein the reaction temperature is within a range of 10 C. to 50 C.

31. The process of claim 16, wherein the amino acid bearing a thiol group or the peptide bearing a thiol group is chosen from cysteine, homocysteine, glutathione and thioredoxin, the organic reducing compound is a hydrogen-donating organic reducing compound bearing a hydroxyl function and is chosen from alcohols, polyols, and sugars, the cofactor common to the reductase enzyme and the dehydrogenase enzyme is a flavinic cofactor or a nicotinic cofactor, and the L-methionine precursor is chosen from O-acetyl-L-homoserine and O-succinyl-L-homoserine.

32. The process of claim 16, wherein the amino acid bearing a thiol group or the peptide bearing a thiol group is glutathione, the organic reducing compound is the organic reducing compound is chosen from glucose, glucose 6-phosphate, and isopropanol, the cofactor common to the reductase enzyme and the dehydrogenase enzyme is a flavinic cofactor or a nicotinic cofactor, and the L-methionine precursor is chosen from O-acetyl-L-homoserine and O-succinyl-L-homoserine.

33. The process of claim 16, wherein the amino acid bearing a thiol group or the peptide bearing a thiol group is glutathione, the organic reducing compound is the organic reducing compound is glucose, the cofactor common to the reductase enzyme and the dehydrogenase enzyme is NADPH, and the L-methionine precursor is O-acetyl-L-homoserine.

Description

EXAMPLE 1: PROCESS IN 2 SUCCESSIVE STEPS

[0090] 10 ml of glutathione enzymatic complex (Aldrich) and 19.2 g (0.1 mol) of glucose are introduced into a reactor R1 containing 150 ml of 0.1 mol/l phosphate buffer at pH 7.30. The solution of enzymatic complex contains: 185 mg (0.6 mmol) of glutathione, 200 U of glutathione reductase, 50 mg (0.06 mmol) of NADPH and 200 U of glucose dehydrogenase. The reaction medium is brought to 25 C. with mechanical stirring. A first sample is taken at t=0. Subsequently, the dimethyl disulfide (9.4 g, 0.1 mol) is placed in a burette and added dropwise to the reactor; the reaction begins. A stream of nitrogen is placed in the reactor.

[0091] Gas chromatography analysis of the gases leaving the reactor shows virtually essentially the presence of nitrogen and methyl mercaptan (some traces of water). These outlet gases are sent into the reactor R2. The DMDS is introduced in 6 hours into the reactor R1. A final gas chromatography analysis of the reaction medium of the reactor R1 confirms the absence of DMDS, and an analysis by UPLC/mass spectrometry shows traces of glucose and the virtually exclusive presence of gluconolactone (traces of gluconic acid).

[0092] In parallel, 5 g of O-acetyl-L-homoserine (OAHS) (the O-acetylhomoserine was synthesized from L-homosenne and acetic anhydride as per Sadamu Nagai, Synthesis of O-acety-L-homoserine, Academic Press, (1971), vol. 17, pp. 423-424) are introduced into the second reactor R2 containing 75 ml of 0.1 mol.Math.l.sup.1 phosphate buffer at pH 6.60. The solution is brought to 35 C. with mechanical stirring.

[0093] Before the reaction starts, a sample (t=0) of 1 ml of the reaction medium is taken. A solution of pyridoxal phosphate (1.6 mmol, 0.4 g) and the enzyme O-acetyl-L-homoserine sulfhydrylase (0.6 g) are dissolved in 10 ml of water then added to the reactor.

[0094] The methyl mercaptan is introduced via the reaction of the reactor R1 and propelled by a nitrogen stream. The reaction then begins. The formation of L-methionine and the disappearance of OAHS are monitored by HPLC. The outlet gases from the reactor R2 are trapped in a 20% aqueous sodium hydroxide solution. The analyses show that the OAHS has been converted to a degree of 52% into L-methionine and that the excess DMDS has been converted into methyl mercaptan found in the sodium hydroxide trap.

EXAMPLE 2: ONE POT PROCESS

[0095] 10 ml of the enzymatic complex, 6 g (33 mmol) of glucose and 5 g (31 mmol) of O-acetyl-L-homoserine (OAHSthe O-acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydride as per Sadamu Nagai, Synthesis of O-acetyl-I-homoserine, Academic Press, (1971), vol. 17, pp. 423-424) are introduced into a reactor containing 150 ml of 0.2 mol.Math.l.sup.1 phosphate buffer at pH 7. The solution of the enzymatic complex contains: 185 mg (0.6 mmol) of glutathione, 200 U of glutathione reductase, 50 mg (0.06 mmol) of NADPH, 200 U of glucose dehydrogenase, 0.4 g (1.6 mmol) of pyridoxal phosphate and 0.6 g of O-acetyl-L-homoserine sulfhydrylase.

[0096] The reaction medium is brought to 27 C. with mechanical stirring. A first sample at t=0 is taken. Subsequently, the dimethyl disulfide (3 g, 32 mmol) is placed in a burette and added dropwise to the reactor which has been closed to avoid any release of methyl mercaptan; the reaction begins. The reaction is monitored by HPLC to see the disappearance of the OAHS and the formation of the L-methionine. After 6 hours, 21% of the OAHS has been converted into L-methionine, demonstrating the possibility of producing L-methionine by a one pot process from an L-methionine precursor, DMDS and an organic reducing compound.